Transcript 06 Classification and modern methods of diagnostics

Microbiology, Virology and Immunology Department
Classification and modern
methods of diagnostics of
infectious diseases
METHODS OF EXAMINATION IN
MICROBIOLOGY
BACTERIOSCOPIC
BACTERIOLOGICAL
SEROLOGICAL
BIOLOGICAL
ALLERGIC
EXPRESS-DIAGNOSIS
BACTERISCOPIC METHOD
Staphylococcus aureus
Escherichia coli
M. tuberculosis
M. avium
BACTERISCOPIC METHOD
Borrelia
Yersinia pestis
Neisseria gonorrhoeae
Corynebacterium diphtheriae
BACTERISCOPIC METHOD
Bacillus anthracis
Clostridium tetani
Clostridium botulinum
BACTERISCOPIC METHOD
BACTERIOLOGICAL
METHOD
Isolation of pure culture
Examples of varous colony morphologies.
The appearance of colonies on a plate is
species specific and can be very helpful in
identifying.
Pure culture of conditionally-pathogenic
microbes may be causative agents of disease
according to the such signs:
 microbes are present in tested material from
pathologic focus in the amount of 104-105 colonyforming units (CFU) in 1 ml or 1 g;
 repeated isolation from the same material the
same culture;
 increasing in the patient’s serum antibodies to
the autostrains or microbial culture, which can be
causative agent.
Necessary rules
before collection of tested material:
 to take material before antibacterial therapy
beginning or after some time after antibiotic
inoculation which is necessary for its excretion
from the organism (as a rule 8-10 hours);
 to take material from infectious focus or examine
proper discharges;
 hold on to the strict aseptic for the purpose to
prevent contamination of the specimen by
microflora of environment;
Necessary rules
before collection of tested material:
 material is taken into the sterile boxes;
clinical specimen with anaerobic bacteria
must be protected from atmosphere oxygen
action;
 the collection of an adequate specimen is
useless if the time between collection and
culturing allows the disease-producing
organism to die (in another cases it’s
necessary to use the refrigerator or special
transport media);
Necessary rules
before collection of tested material:
isolation of viruses, Rickettsia, Chlamidia
is made in specialize laboratories;
to clinical specimen a proper document is
added, which has data , which has data for
correct microbiological examination.
BACTERIOLOGICAL METHOD
Most specimens received in a clinical microbiology lab are plated
onto Blood Agar
Gold’s streak
BACTERIOLOGICAL METHOD
Bacillus
Proteus
BACTERIOLOGICAL METHOD
Staphylococcus
Streptococcus
BACTERIOLOGICAL METHOD
Klebsiella pneumoniae
Mannitol Salt Medium
Most non-pathogenic
staphylococci will not ferment
mannitol
BACTERIOLOGICAL METHOD
isolation of pure culture
BACTERIOLOGICAL METHOD
biochemical identification
Biochemical Reactions on API Strip
Proteus mirabilis
Escherichia coli
Positive
Lactose Positive Colonies
MacConkey Agar
Negative
Indole Reactions
BACTERIOLOGICAL METHOD
Serological method
Agglutination test
BACTERIOLOGICAL METHOD
Serological method
Agglutination test
BACTERIOLOGICAL METHOD
Serological method
IHT
BACTERIOLOGICAL METHOD
Serological method
Ring precipitation test
BACTERIOLOGICAL METHOD
Serological method
Doublel Immune Diffusion (Ouchterlony’) Test
BACTERIOLOGICAL METHOD
Serological method
Radial Immune Diffusion (Mancini’s) Test
BACTERIOLOGICAL METHOD
Serological method
CFT
BACTERIOLOGICAL METHOD
Serological method
ELISA
SEROLOGICAL METHODS
Express-diagnosis
Treponema pallidum
Bordetella pertussis
IFA
Molecular Diagnostics
Immunological Diagnostics Methods
Immunofluorescence
Protein A has the ability to bind to IgG
Fluorochromes
-Fluorescein (490→517nm)
-Rhodamine (515→546nm)
-Phycoerythrin
mIgM-producing B cells indirectly stained with
rhodamine-conjurated secondary Ab under a
fluorescence microscope.
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BIOLOGICAL METHOD
tetanus
botulism
BIOLOGICAL METHOD
TBC in rabbit
TBC in guinea pig
ALLERGIC METHOD
Mantoux’s test
Applications of Molecular Based
Testing in Clinical Microbiology
 Rapid or high-throughput identification of
microorganisms
 Detection and analysis of resistance genes
 Genotyping
 Classification
 Discovery of new microorganisms
Specimen Collection
 Preserve viability/nucleic acid integrity of target
microorganisms
 Avoid contamination
 Appropriate time and site of collection (blood,
urine, other)
 Use proper equipment (coagulant, wood, or
plastic swab shafts)
 Commercial collection kits are available
 The Clinical and Laboratory Standards Institute
(CLSI) has guidelines for proper specimen
handling
Sample Preparation
 Consider the specimen type (stool, plasma, CSF)
 More rigorous lysis procedures are required to
penetrate cell walls
 Consider the number of organisms in the sample
 Inactivate inhibitors (acidic polysaccharides in
sputum or polymerase inhibitors in CSF)
 Inactivate RNases
Molecular Diagnostics
Immunological Diagnostics Methods
ELISA
•
Addition of a specific antibody (primary
antibody) which will bind to the test molecule
if it is present.
•
Washing to remove unbound molecules.
•
Addition of secondary antibody which will
bind to the primary antibody.
•
The secondary antibody usually has attached
to it an enzyme e.g. alkaline phosphatase.
•
Wash to remove unbound antibody.
•
Addition of a colourless substrate which will
react with the secondary antibody to give a
colour reaction which indicates a positive
result.
-> can be used for quasi High-throughput!!!
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Molecular Diagnostics
Immunological Diagnostics Methods
ELISA -Variants
Detection based on enzyme
catalyzed reactions:
1.alkaline ⓟ
2.horseradish peroxidase
3. β-galactosidase
Detection based on
fluorescent labeled
secondary antibody
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Molecular Diagnostics
Immunological Diagnostics Methods
Western blot
SDS-Page: separates the components according to
their molecular weight.
Blot: the proteins in the gel are transferred to the
sheet of nitrocellulose or nylon by the passage
of an electric current.
Immunoreaction: probed with Ab & then radiolabeled or
enzyme-linked 2nd Ab.
Detection: a position is visualized by means of an ELISA
reaction.
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Molecular Diagnostics
DNA Diagnostic Systems
PCR based methods
-> The presence of the appropriate amplified size fragment confirms the presence of the
target.
-> Specific primers are now available for the detection of many pathogens including bacteria
(E. coli, M. tuberculosis), viruses (HIV) and fungi.
Example: Using PCR to Detect for HIV
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RT-PCR (reverse transcriptase PCR).
HIV has a ssRNA genome.
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Other examples:
-> Using PCR to Detect DMD deletions (60% of
mutations are deletions)
•
Specific primers are used to
amplify a 156 bp portion of the
HIV gag gene.
Using standards the amount of
PCR product can be used to
determine the viral load.
PCR can also be used as a
prognostic tool to determine
viral load.
This method can also be used to
determine the effectiveness
antiviral therapy.
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Polymerase Chain Reaction
Capable of amplifying tiny quantities of nucleic acid.
Cells separated and lysed.
Double stranded DNA separated into single strands.
Primers, small segments of DNA no more than 20-30 nucleotides
long added.
 Primers are complementary to segments of opposite strands of
that flank the target sequence.
 Only the segments of target DNA between the primers will be
replicated.
 Each cycle of PCR consists of three cycles:
 denaturation of target DNA to separate 2 strands.
 annealing step in which the reaction mix is cooled to allow primers to
anneal to target sequence
 Extension reaction in which primers initiate DNA synthesis using a
DNA polymerase.
 These three steps constitute a thermal cycle
 Each PCR cycle results in a doubling of target sequences and
typically allowed to run through 30 cycles, one cylce takes
approximately 60-90 seconds.
(95°C)
(55°C-65°C).
(72°C)
The final product can be checked on an agarsoe gel to make sure it has correct
size and can be sequenced.
The final product is available in high conc. and can be used for cloning (gene that
has a product), diagnosis (gene of a virus) or fingerprinting (forensic
investigation in crime scenes).
PCR Polymerase Chain Reaction
Continue
30 cycles
Denature
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PCR tubes
Agarose gel to detect the products
Thermocycler
Sequencing
Gel Electrophoresis apparatus
Loading the gel
After reaction is complete, the DNA is loaded into an agarose gel, in order to
visualize the bands.
The DNA is mixed with Glycerol and specific reagents when loading into the gel.
An electric current is applied so the DNA moves towards the + ve electrode
according to it s size
Small fragments move faster and reach the end of the gel, larger fragments move
slower and are at the beginning the gel.
Gel is stained to view the DNA bands.
Larger bands up
Direction of migration
Smaller bands down
Agarose gel stained with Ethedium bromide and visualized under UV
Applications of PCR
In forensic medicine:
For instance, trace amounts of DNA, in fluids such as blood
or semen or in tissue such as hair, can be amplified by
PCR and analyzed to see whether the DNA is identical to
that of a person suspected of committing a crime.
In clinical diagnosis:
The technique enabled clinicians to detect infection by the
AIDS virus when other methods have failed, in addition to
other diseases.
Diagnosis genetic diseases, such as sickle cell anemia in
fetus still in its mother’s uterus, by amplifying the genetic
information provided by just a few fetal cells, which can
be obtained without harming the fetus.
Target Microorganisms for MolecularBased Testing
 Those that are difficult or time-consuming to
isolate
 e.g., Mycobacteria
 Hazardous organisms
 e.g., Histoplasma, Coccidiodes
 Those without reliable testing methods
 e.g., HIV, HCV
 High-volume tests
 e.g., S. pyogenes, N. gonorrhoeae, C. trachomatis